Therapeutic angiogenesis using genetically engineered human endothelial cells.

Cell therapy holds promise as a method for the treatment of ischemic disease. However, one significant challenge to the efficacy of cell therapy is poor cell survival in vivo. Here we describe a non-viral, gene therapy approach to improve the survival and engraftment of cells transplanted into ischemic tissue. We have developed biodegradable poly(ß-amino esters) (PBAE) nanoparticles as vehicles to genetically modify human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF). VEGF transfection using these nanoparticles significantly enhanced VEGF expression in HUVECs, compared with a commercially-available transfection reagent. Transfection resulted in the upregulation of survival factors, and improved viability under simulated ischemic conditions. In a mouse model of hindlimb ischemia, VEGF nanoparticle transfection promoted engraftment of HUVECs into mouse vasculature as well as survival of transplanted HUVECs in ischemic tissues, leading to improved angiogenesis and ischemic limb salvage. This study demonstrates that biodegradable polymer nanoparticles may provide a safe and effective method for genetic engineering of endothelial cells to enhance therapeutic angiogenesis.

Genetic engineering of human stem cells for enhanced angiogenesis using biodegradable polymeric nanoparticles.

Stem cells hold great potential as cell-based therapies to promote vascularization and tissue regeneration. However, the use of stem cells alone to promote angiogenesis remains limited because of insufficient expression of angiogenic factors and low cell viability after transplantation. Here, we have developed vascular endothelial growth factor (VEGF) high-expressing, transiently modified stem cells for the purposes of promoting angiogenesis. Nonviral, biodegradable polymeric nanoparticles were developed to deliver hVEGF gene to human mesenchymal stem cells (hMSCs) and human embryonic stem cell-derived cells (hESdCs). Treated stem cells demonstrated markedly enhanced hVEGF production, cell viability, and engraftment into target tissues. S.c. implantation of scaffolds seeded with VEGF-expressing stem cells (hMSCs and hESdCs) led to 2- to 4-fold-higher vessel densities 2 weeks after implantation, compared with control cells or cells transfected with VEGF by using Lipofectamine 2000, a leading commercial reagent. Four weeks after intramuscular injection into mouse ischemic hindlimbs, genetically modified hMSCs substantially enhanced angiogenesis and limb salvage while reducing muscle degeneration and tissue fibrosis. These results indicate that stem cells engineered with biodegradable polymer nanoparticles may be therapeutic tools for vascularizing tissue constructs and treating ischemic disease.

Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation.

RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-alpha (TNF-alpha). siRNA was designed and synthesized targeting tumor necrosis factor-alpha receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-alpha expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-alpha expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model.

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.

Regeneration of whole meniscus using meniscal cells and polymer scaffolds in a rabbit total meniscectomy model.

The current treatments of meniscal lesion in knee joint are not perfect to prevent adverse effects of meniscus injury. Tissue engineering of meniscus using meniscal cells and polymer scaffolds could be an alternative option to treat meniscus injury. This study reports on the regeneration of whole medial meniscus in a rabbit total meniscectomy model using the tissue engineering technique. Biodegradable scaffolds in a meniscal shape were fabricated from polyglycolic acid (PGA) fiber meshes that were mechanically reinforced by bonding PGA fibers at cross points with 75:25 poly(lactic-co-glycolic acid). The compressive modulus of the bonded PGA scaffold was 28-fold higher than that of nonbonded scaffold. Allogeneic meniscal cells were isolated from rabbit meniscus biopsy and cultured in vitro. The expanded meniscal cells were seeded onto the polymer scaffolds, cultured in vitro for 1 week, and transplanted to rabbit knee joints from which medial menisci were removed. Ten or 36 weeks after transplantation, the implants formed neomenisci with the original scaffold shape maintained approximately. Hematoxylin and eosin staining of the sections of the neomenisci at 6 and 10 weeks revealed the regeneration of fibrocartilage. Safranin-O staining showed that abundant proteoglycan was present in the neomenisci at 10 weeks. Masson's trichrome staining indicated the presence of collagen. Immunohistochemical analysis showed that the presence of type I and II collagen in neomenisci at 10 weeks was similar to that of normal meniscal tissue. Biochemical and biomechanical analyses of the tissue-engineered menisci at 36 weeks were performed to determine the quality of the tissue-engineered menisci. Tissue-engineered meniscus showed differences in collagen content and aggregate modulus in comparison with native meniscus. This study demonstrates, for the first time, the feasibility of regenerating whole meniscal cartilage in a rabbit total meniscectomy model using the tissue engineering method.